TY - JOUR
T1 - Insights into the quality of DnaA boxes and their cooperativity
AU - Hansen, Flemming G.
AU - Christensen, Bjarke Bak
AU - Nielsen, Christina Bang
AU - Atlung, Tove
PY - 2006
Y1 - 2006
N2 - Plasmids carrying the mioC promoter region with its two DnaA boxes are as efficient in titration of DnaA protein as plasmids carrying a replicationinactivated oriC region with its five DnaA boxes. The two DnaA boxes upstream of the mioC promoter were mutated in various ways to study the cooperativity between the DnaA boxes, and to study in vivo the in vitrodefined 9mer DnaA box consensus sequence TTA/TTNCACA). The quality and cooperativity of the DnaA oxes were determined in two complementary ways: as titration of DnaA protein leading to derepression of the dnaA promoter, and as repression of the mioC promoter caused by the DnaA protein binding to the DnaA boxes. Titration of DnaA protein correlated with repression of the mioC promoter. The level of titration and repression with the normal promoter-proximal box (TTTTCCACA) depends strongly on the presence and the quality of a DnaA box in the promoter-distal position, whereas a promoter-proximal DnaA box with the sequence TTATCCACA titrated DnaA protein and caused significant repression of the mioC promoter without a promoter-distal DnaA box. The quality of the eight different consensus DnaA boxes located in the
promoter-proximal position was determined: TTATCCACA had the
highest affinity for DnaA protein. In the third position, A was better than T, and the four possibilities in the fifth position could be ranked as C>A>=G>T. Parallel in vitro experiments using a purified DNA-binding domain of DnaA protein gave the same ranking of the binding affinities of the eight DnaA boxes.
AB - Plasmids carrying the mioC promoter region with its two DnaA boxes are as efficient in titration of DnaA protein as plasmids carrying a replicationinactivated oriC region with its five DnaA boxes. The two DnaA boxes upstream of the mioC promoter were mutated in various ways to study the cooperativity between the DnaA boxes, and to study in vivo the in vitrodefined 9mer DnaA box consensus sequence TTA/TTNCACA). The quality and cooperativity of the DnaA oxes were determined in two complementary ways: as titration of DnaA protein leading to derepression of the dnaA promoter, and as repression of the mioC promoter caused by the DnaA protein binding to the DnaA boxes. Titration of DnaA protein correlated with repression of the mioC promoter. The level of titration and repression with the normal promoter-proximal box (TTTTCCACA) depends strongly on the presence and the quality of a DnaA box in the promoter-distal position, whereas a promoter-proximal DnaA box with the sequence TTATCCACA titrated DnaA protein and caused significant repression of the mioC promoter without a promoter-distal DnaA box. The quality of the eight different consensus DnaA boxes located in the
promoter-proximal position was determined: TTATCCACA had the
highest affinity for DnaA protein. In the third position, A was better than T, and the four possibilities in the fifth position could be ranked as C>A>=G>T. Parallel in vitro experiments using a purified DNA-binding domain of DnaA protein gave the same ranking of the binding affinities of the eight DnaA boxes.
U2 - 10.1016/j.jmb.2005.10.036
DO - 10.1016/j.jmb.2005.10.036
M3 - Journal article
SN - 0022-2836
VL - 355
SP - 85
EP - 95
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -